Arf6 KO platelets are defective in Fg uptake in vivo and ex vivo. (A) Arf6 deletion impairs platelet uptake of Fg in vivo. (i) WT and KO mice were injected with biotin-Fg via the retro-orbital sinus. Platelets were harvested 24 hours postinjection. Platelet extracts (1 × 107/lane) were loaded, and the indicated proteins were probed for by western blotting using corresponding antibodies. Each lane represents platelets from a single mouse. A mouse without injection (No Inj) was included as a negative control. β-Actin was used as a loading control. (ii) Quantification of biotin-Fg levels in panel Ai was performed using ImageQuantTL and analyzed by SigmaPlot 12.0. (B) Arf6 deletion impairs Fg uptake by platelets ex vivo. (i) Washed platelets (5 × 108/mL) from WT and KO mice were incubated with FITC-Fg (0.05 mg/mL) for 1 hour at 37°C. After removing the extracellular FITC-Fg, platelets were incubated with HEPES Tyrode buffer (pH = 7.4) for another 2 hours. Platelets were fixed with 2% paraformaldehyde overnight at 4°C and subjected to epifluorescence microscopy. The extracellular signal was quenched with 0.1% trypan blue, and representative images, from 4 experiments, are presented. (ii) The number of FITC-positive puncta per platelet was manually counted, and platelets were grouped according to that number. The percentage platelets in each group relative to total was calculated and plotted as a bar graph. Statistical significance was determined using rank sum test. (C) Overexposure of biotin blot in panel Ai, to probe for fragments of biotin-Fg. Starting biotin-Fg (Pre Inj) was included as a comparison. (D) Arf6 deletion impedes uptake of FITC-Fg ex vivo. Washed platelets (1.0 × 109/mL) from 3 WT (black) and 3 KO (open) mice were separately incubated with FITC-Fg (0.15 mg/mL) at 37°C for the indicated times and then fixed with 2% paraformaldehyde overnight at 4°C. Intracellular FITC-Fg levels were measured by flow cytometry, after addition of trypan blue, and expressed as GMFI (mean ± standard deviation). Statistical significance was determined by Student t test. (E) Megakaryocyte uptake of FITC-Fg is below detection under the conditions used. WT and KO mice were injected with FITC-Fg (0.75 mg/mouse) via the retro-orbital sinus. Each group included 2 mice. Bone marrow cells and platelets were harvested 24 hours postinjection and fixed with 2% paraformaldehyde. DIC images and fluorescence images are presented. Megakaryocytes were identified by CD41 staining. (i) The exposure time for FITC-Fg and CD41 images is 2 seconds and 0.5 seconds, respectively. The scale bar is 10 µm. (ii) The exposure time for FITC-Fg is 1 second. The scale bar represents 5 µm. Representative images were selected from >10 different fields of each group.
